Alphaviruses are arthropod-transmitted viruses that cause medically-important human infections worldwide. Specific therapeutics for these viruses are lacking, and only experimental vaccines are available for these infections. In this Developmental/Exploratory Grant, we will generate and characterize in detail human anti- alphavirus antibodies, filling an unaddressed gap in our knowledge regarding the human antibody repertoire to alphavirus infection. Although past studies have characterized antibody responses to prototypical alphaviruses, such as Sindbis virus, in mice; little is known regarding the specific epitopes or the repertoire of human antibodies to natural alphavirus infection. We propose to use human hybridoma technology that has proven capable of generating potent neutralizing antibodies to each of the three 20th century pandemic viruses, despite the fact that each of these viruses circulated many years ago, to generate human antibodies to two medically important alphaviruses, Venezuelan equine encephalitis (VEE) and Mayaro (MAY) virus. We will obtain blood from a unique population of individuals from endemic areas of alphavirus transmission in Peru who have been definitively diagnosed with recent VEE or MAY virus infection. From the B cells of these individuals, we will generate human monoclonal antibodies that neutralize VEE and MAY viruses, and we will characterize in vitro and in vivo the human monoclonal antibodies. VEE and MAY viruses cause significant morbidity in Latin American countries and also pose threats to the United States. VEE virus alone accounts for as much as 7% of the supposed dengue fever cases in certain tropical regions and is considered a NIAID Category B priority pathogen whereas MAY virus causes recurrent incapacitating arthralgias that can last several months, similar to Chikungunya virus. Completion of this study will generate the first naturally- occurring human antibodies to VEE and MAY virus, providing insight into the antibody repertoire toward these important human pathogens. It will also seek to identify VEE and MAY virus-specific and more broadly cross- reactive (to other alphaviruses) antibodies and their epitopes. Such antibodies are potential therapeutics, and information regarding their epitopes, particularly cross-reactive epitopes, may suggest optimized vaccine approaches that may successfully target multiple alphaviruses.